We have discovered a series of heme proteins in the saliva of the blood- sucking insect Rhodnius prolixus that act as vasodilators and anti-platelet aggregation agents when injected into victim. These heme proteins are novel fe(III) proteins that are reversible carriers of nitric oxide (NO). Because of their NO-carrying function, these proteins have been named nitrophorins," and we have characterized them in enough detail to show that they are worthy of much more extensive investigation. We have also partially characterized a nitrophorin from the saliva of the bedbug, Cimex lectularius, suggesting that salivary nitrophorins may be a fairly common mechanism by which insects are assured of obtaining a blood meal. Such insect salivary nitrophorins are the only known reversible NO-carrying proteins. Our goal is to extensively characterize them, in terms of their physical properties and chemical reactivity, in order to achieve a better understanding of the interaction of NO with heme proteins, to provide insight concerning other NO-binding hemeproteins and the general factors that mediate the reactivity of coordinated NO, and to provide perspective on the biochemical, physiological, and pharmacological roles that these interactions play in human health. For the first grant period our goals for characterization of the nitrophorins are: (1) The nitrophorins from R. prolixus will be characterized as a function of pH by vibrational spectroscopic technique (infrared and resonance Raman spectroscopy). (2) The kinetics of NO binding and release, and possible nitrosation reaction of the nitrophorins and relevant model heme complexes, will be investigated as a function of pH by stopped flow and nanosecond laser flash photolysis. (3) The nitrophorins will be examined in the presence an absence of No and histamine by EPR spectroscopy. The NO complex of the dithionite-reduced nitrophorins will be carefully analyzed y EPR spectroscopy in an attempt to obtain definitive evidence of the sixth ligand bound to the heme. (4) The nitrophorins will be characterized by magnetic circular dichroism (MCD) spectroscopy in the native NO-bound state, as well as the high-pH NO-free form and the histamine complex. The dithionite-reduced state, in the absence and presence of NO will also be characterized. (5) The nitrophorins will be characterized by Mossbauer spectroscopy in all appropriate spin, ligation and oxidation states. Relevant model heme analogs of each of these forms will be investigated as well, in order to obtain base line information as to the importance of porphyrin and axial ligand electronic effects, ligand steric effects, and imidazole deprotonation effects on Mossbauer parameters. (6) The nitrophorins will be characterized in detail by NMR spectroscopy as a function of pH, heme axial ligand, and temperature, in order to obtain detailed information concerning the magnetic environment of the heme, axial ligand binding characteristics, and the residues lining the heme pocket. (7) Electarochemical investigations of the NO-bound nitrophorins, metmyoglobin, and appropriate model heme complexes will be carried out in order to determine what differences exist in the redox chemistry of the salivary heme proteins of R. prolixus, and why they differ in their No- binding characteristics from methemoglobin and metmyoglobin, both of which readily autoreduce in the presence of excess No. (8) X-ray crystallographic investigations of the most abundant nitrophorin, NP1, will be carried out in order to determine the complete three-dimensional structure of the NO-bound and NO-free (high pH) forms of this protein. (9) A series of water-soluble model hemes will be synthesized and investigated by each of the above-mentioned techniques, in order to allow testing hypotheses concerning the factors that affect the pH dependence of NO binding and release.